A sequence listing appendix is submitted under 1.821(c) and is incorporated herein by reference.
1. Field of the Invention
The present invention relates generally to molecular biology and biochemistry and more specifically to a coactivator protein, p/CIP, which is involved in regulating gene expression by CBP/p300-dependent transcription factors, and to methods of using the coactivator protein to selectively regulate gene expression.
2. Background Information
Regulation of gene expression is mediated by sequence-specific transcription factors that bind to target genes and activate or repress transcription. Many of these factors are controlled by extracellular signals that switch the factors between inactive and active states. Such signals can result in post-translational modification as observed, for example, with the members of the STAT family of transcription factors, or can result in ligand-induced conformational changes as observed, for example, with members of the nuclear receptor family of transcription factors.
Coactivator proteins have been identified that are recruited to the active forms of such transcription factors and are required for their transcriptional effects. The coactivators, CBP and p300, for example, serve essential roles in transcriptional activation by several classes of regulated transcription factors, including nuclear receptors, STAT factors, AP-1 proteins, NF-KB and CREB. In addition, a more recently discovered family of proteins, termed nuclear receptor coactivator (NCoA) proteins, can interact with various nuclear receptors in a ligand-dependent manner and also can interact with CBP and p300.
Two members of the NCoA family of proteins, NCoA-1 and NCoA-2, appear to have relatively selective roles in mediating the transcriptional effects of nuclear receptors. Evidence indicates, however, that additional factors are required for the transcriptional activities of many CBP-dependent transcription factors, including STAT 1, AP-1 and CREB, and that complexes containing such coactivators, for example, CBP/p300 and NCoA, are involved in transmitting an activation signal to the promoter. Since CBP and p300-containing complexes appear to be limiting in cells, antagonistic interactions between signaling pathways can be due, at least in part, to competition for these complexes. Thus, a need exists to identify different classes of transcription factors that are regulated by a CBP-containing complex and to identify the coactivator proteins involved in such complexes. The present invention satisfies this need and provides related advantages as well.
The present invention provides a substantially purified nucleic acid molecule having a nucleotide sequence encoding a transcriptional coactivator protein, designated p/CIP, which binds to CBP/p300-dependent transcription factors and regulates their activity. For example, the invention provides a substantially purified nucleic acid molecule having the nucleotide sequence shown in FIG. 1, which encodes murine p/CIP, and a nucleotide sequence complementary to that shown in FIG. 1.
The invention also provides a substantially purified nucleic acid molecule encoding an active fragment of a p/CIP polypeptide, which has a nucleotide sequence encoding substantially the same amino acid sequence as a portion of a p/CIP polypeptide. Such a nucleic acid molecule can encode, for example, an active fragment including a CBP interaction domain, such as a fragment having about amino acids 758 to 1115 of p/CIP shown in FIG. 1, or a nuclear receptor interaction domain, such as a fragment having about amino acids 591 to 803 or about amino acids 680 to 740 of p/CIP shown in FIG. 1.
Further provided herein is a substantially purified nucleic acid molecule having a nucleotide sequence encoding a full length mouse NCoA-2 protein, which is related to p/CIP. The invention also provides a substantially purified NCoA-2 active fragment, having a nucleotide sequence encoding substantially the same amino acid sequence as a portion of a NCoA-2 polypeptide. Such a NCoA-2 active fragment can include, for example, a nuclear receptor interaction domain.
The invention also provides vectors comprising a nucleic acid molecule of the invention and host cells containing such vectors. In addition, the invention provides a substantially purified p/CIP nucleotide sequence having at least about 14 consecutive nucleotides of the nucleotide sequence shown in FIG. 1, or a nucleotide sequence complementary thereto.
The present invention also provides a substantially purified p/CIP polypeptide, which can bind to a CBP/p300-dependent transcription factor and regulate its activity. For example, the invention provides a substantially purified p/CIP polypeptide having substantially the same amino acid sequence as p/CIP shown FIG. 1. The invention additionally provides a substantially purified p/CIP active fragment having substantially the same amino acid sequence as a portion of a p/CIP polypeptide. A particularly useful p/CIP active fragment can include, for example, a CBP interaction domain or a nuclear receptor interaction domain, or can be an portion of a p/CIP polypeptide useful for eliciting production of an antibody that specifically binds to p/CIP.
The invention further provides a substantially purified NCoA-2 polypeptide having substantially the same amino acid sequence as amino acid sequence shown in FIG. 2a. Active fragments of a NCoA-2 polypeptide of the invention also are provided herein.
The invention also provides anti-p/CIP antibodies that specifically bind to p/CIP, as well as p/CIP-binding fragments of such antibodies. The invention further provides anti-NCoA-2 antibodies and antigen binding fragments thereof. In addition, the invention provides cell lines producing anti-p/CIP antibodies or anti-NCoA-2 antibodies.
The present invention further provides methods of identifying an effective agent that alters the association of p/CIP or NCoA-2 polypeptide with a second protein, such as a nuclear receptor or a CBP, which associates with the p/CIP or NCoA-2 polypeptide in vitro or in vivo. The method includes the steps of contacting a p/CIP or NCoA-2 polypeptide with an agent under conditions that allow the p/CIP or NCoA-2 polypeptide to associate with the second protein, and detecting an altered association of the p/CIP or NCoA-2 polypeptide with the second protein. An agent that alters the association of p/CIP, for example, with a second protein can be a peptide, a polypeptide, a peptidomimetic or an organic molecule, such an effective agent being useful, for example, for modulating the level of transcription in a cell. For example, a peptide portion of p/CIP comprising a helical leucine-rich, charged domain (LCD), can inhibit the transcriptional activity of one type of nuclear receptor, such as the retinoic acid receptor, but not of a second, related nuclear receptor such as the estrogen receptor, whereas a second LCD of p/CIP can inhibit signal transduction induced by interferon xcex3, but not signal transduction induced by retinoic acid. Thus, selected peptide portions of p/CIP or of NCoA-2 can be valuable for regulating gene expression in a cell, and these and other effective agents can have therapeutic efficacy.